Proteome changes associated with Leishmania donovani promastigote adaptation to oxidative and nitrosative stresses

Phosphorylation of Translation Initiation Factor 2-Alpha in Leishmania donovani under Stress Is Necessary for Parasite Survival

“…We analyzed intracellular ROS generation in promastigotes using 2=,7=-dichlorodihydrofluorescein diacetate (H 2 DCFDA) dye. The ROS level for L. donovani promastigotes exposed to the higher dose of 70 M SAG (SAG 70 ) was found to be ϳ1.2-fold higher (P Ͻ 0.05) than that for promastigotes exposed to the lower dose (SAG 40 ). Similarly, for amphotericin B treatment, promastigotes subjected to the higher dose of Amp B (Amp B 0.06 ) showed ϳ1.3-fold-higher (P Ͻ 0.05) ROS accumulation than did promastigotes subjected to the lower dose of Amp B (Amp B 0.03 ).…”

“…As results from previous experiments showed that drug-generated ROS are also capable of inducing LdeIF2␣ phosphorylation, we estimated parasite viability under drug pressure in the presence of GSK2606414. The percent viabilities of promastigotes after 48 h of exposure to SAG 40 or SAG 70 were 71% and 59%, respectively, whereas in the presence of GSK, viabilities were 62% and 46%, respectively. Similarly, with exposure to amphotericin B, viz., Amp B 0.03 or Amp B 0.06 , the percent viabilities were 76% and 52%, respectively, which were further decreased significantly in the presence of GSK2606414 to 61% for treatment with Amp B 0.03 plus GSK and to 39% for treatment with Amp B 0.06 plus GSK (Fig.…”

“…(E) Percent cell viability of L. donovani promastigotes subjected to oxidative stress generated due to H 2 O 2 treatment or in the presence of GSK2606414 and under H 2 O 2 stress along with GSK2606414 after 4 h of treatment. (F) Percent viability at 48 h of promastigotes exposed to SAG 40 …”

“…Overall, we conclude that stress-induced phosphorylation of LdeIF2␣ is a crucial event in the life cycle of L. donovani through (i) transformation of the motile promastigote form into the nonmotile amastigote form; (ii) decreased global translation and upregulation of stress-responsive genes during transformation or in the amastigote form, which indicates the adaptive response of parasites to conserve energy and divert it toward the minimum essential pathways required for survival, exemplified by thiol redox metabolism (27,40); and (iii) use of the pentose phosphate pathway (41) for coping with oxidative stress and energy generation (37), respectively, while converting the more active form into the low-metabolic amastigote form. ROS accumulation, decreased viability, and a drastic decrease in the infectivity rate upon the inhibition of stress-induced LdeIF2␣ phosphorylation emphasize the importance of this stressresponsive pathway.…”

“…For in vitro generation of oxidative stress, H 2 O 2 (200 M) (Merck GmbH, Darmstadt, Germany) was added to the promastigote culture, suspended in fresh medium, and incubated at 25°C inside a basic oxygen demand (BOD) incubator (41). Due to the short half-life of H 2 O 2 , the treatment time was limited to 4 h. SAG and Amp B treatments were given for 8 h at two different doses, viz., 40 M (SAG 40 ) and 70 M (SAG 70 ) for SAG and 0.060 M (Amp B 0.06 ) and 0.030 M (Amp B 0.03 ) for amphotericin B, based on the 50% lethal doses (LD 50 s) of the drugs, as described previously (44). The treatment time for deducing LD 50 s was kept well below 48 h to ensure that parasite viability is not significantly affected upon treatment.…”